T-cell reactivity platform

Abstract

Methods for assaying antigen-specific T-cell activation in vitro, comprising the steps of (a) providing a phagocytable particle, having a candidate antigen polypeptide tightly associated thereto, wherein the particle with the associated polypeptide has been subjected to a denaturing wash resulting in an endotoxin level low enough to not interfere with the subsequent steps; (b) providing a viable antigen-presenting cell; (c) contacting the washed particle with the antigen-presenting cell under conditions allowing phagocytosis of the particle by the antigen-presenting cell; (d) providing a T-cell sample to be assayed comprising viable T-cells; (e) contacting the T-cell sample with the antigen-presenting cell contacted with the particle under conditions allowing specific activation of T-cells in response to an antigen presented by an antigen-presenting cell; and (f) determining the degree of T-cell activation in the T-cell sample.

The invention claimed is: 
     
       1. A method for assaying antigen-specific T-cell activation, comprising the steps of:
 a. providing a phagocytable particle, having a candidate antigen polypeptide associated by covalent linking, wherein the particle with the associated polypeptide has been subjected to a denaturing wash resulting in an endotoxin level low enough to not interfere with steps b-f; 
 b. providing a viable antigen-presenting cell; 
 c. contacting the washed particle with the antigen-presenting cell in vitro under conditions allowing phagocytosis of the particle by the antigen-presenting cell; 
 d. providing a T-cell sample to be assayed, the T-cell sample comprising viable T-cells; 
 e. contacting the T-cell sample with the antigen-presenting cell contacted with the particle in vitro under conditions allowing specific activation of T-cells in response to an antigen presented by an antigen-presenting cell; and 
 f. determining the degree of T-cell activation in the T-cell sample. 
 
     
     
       2. A method for assaying antigen-specific T-cell activation, comprising the steps of:
 a. providing a phagocytable particle; 
 b. associating by covalent linking a candidate antigen polypeptide to the particle; 
 c. subjecting the particle with the associated polypeptide to a denaturing wash resulting in an endotoxin level low enough to not interfere with steps d-h; 
 d. providing a viable antigen-presenting cell; 
 e. contacting the washed particle with the antigen-presenting cell in vitro under conditions allowing phagocytosis of the particle by the antigen-presenting cell; 
 f. providing a T-cell sample to be assayed, the T-cell sample comprising viable T-cells; 
 g. contacting the T-cell sample with the antigen-presenting cell contacted with the particle in vitro under conditions allowing specific activation of T-cells in response to an antigen presented by an antigen-presenting cell; and 
 h. determining the degree of T-cell activation in the T-cell sample. 
 
     
     
       3. The method according to  claim 1 , further comprising the step of comparing the degree of T-cell activation to a relevant reference, whereby a higher degree of T-cell activation in the sample compared to the reference indicates that the candidate antigen results in antigen-specific T-cell activation in the sample. 
     
     
       4. The method according to  claim 1 , wherein determining the degree of T-cell activation in the T-cell sample comprises determining the fraction of activated T-cells in the sample. 
     
     
       5. The method according to  claim 1 , wherein several candidate antigens are assayed against the same T-cell sample. 
     
     
       6. The method according to  claim 5 , wherein at least 10 candidate antigens are assayed against the same T-cell sample. 
     
     
       7. The method according to  claim 1 , wherein the antigen-presenting cell and the T-cell sample are derived from a single individual. 
     
     
       8. The method according to  claim 7 , wherein the antigen-presenting cell and the T-cell sample are derived from the same blood sample from the individual. 
     
     
       9. The method according to  claim 7 , wherein the antigen-presenting cell and the T cell sample are derived from a peripheral blood mononuclear cell (PBMC) sample from the individual. 
     
     
       10. The method according to  claim 1 , wherein the T-cell sample is derived from a tumour. 
     
     
       11. The method according to  claim 1 , wherein the T-cell sample is derived from ascites. 
     
     
       12. The method according to  claim 1 , wherein the T-cell sample comprises CD4 + T-cells, CD8 + T-cells, or both. 
     
     
       13. The method according to  claim 1 , wherein the washed particle, the antigen presenting cell and the T-cell sample are contacted concurrently. 
     
     
       14. The method according to  claim 1 , wherein determining the degree of T-cell activation in the T-cell sample comprises determining the fraction of T-cells contacted with the antigen-presenting cell responding by secretion of IFN-γ, IL-17, IL-22, or combinations thereof. 
     
     
       15. The method according to  claim 1 , wherein determining the degree of T-cell activation in the T-cell sample comprises determining the fraction of T-cells contacted with the antigen-presenting cell responding by secretion of IL-17, IL-22, or both. 
     
     
       16. The method according to  claim 1 , wherein the candidate antigen polypeptide comprises at least 50 amino acids. 
     
     
       17. The method according to  claim 1 , wherein the T-cell sample is from a human and the candidate antigen polypeptide sequence is derived from a human. 
     
     
       18. The method according to  claim 1 , wherein the candidate antigen polypeptide is derived from a polypeptide:
 a. known to be highly expressed in a tissue or cell affected in an autoimmune disease; 
 b. known to be associated with a neoplastic disease; 
 c. known to be associated with an autoimmune disease; 
 d. known to be associated with an infectious disease; or 
 e. known to be associated with allergy or similar hypersensitivity. 
 
     
     
       19. The method according to  claim 1 , comprising the steps of:
 a. providing a phagocytable particle, having a candidate peptide auto-antigen associated by covalently linking, wherein the particle with the associated polypeptide has been subjected to a denaturing wash resulting in an endotoxin level low enough to not interfere with steps b-e; 
 b. providing a peripheral blood mononuclear cell (PBMC) sample from a test subject, the PBMC sample comprising viable T-cells and viable antigen-presenting cells; 
 c. contacting the PBMC sample with the particle in vitro under conditions allowing phagocytosis of the particle by an antigen-presenting cell and allowing specific activation of T-cells in response to an antigen presented by an antigen-presenting cell; 
 d. quantitating the fraction of activated T-cells in the contacted PBMC sample; and 
 e. comparing the quantitated fraction to a comparable quantitated fraction from a healthy subject, wherein a higher fraction of activated T-cells in the test subject as compared to the healthy subject is indicative of autoreactivity against the candidate auto-antigen in the test subject.